Modified antibodies and related compounds, compositions, and methods of use

ABSTRACT

Provided herein are modified antibodies, compounds used to make them, and intermediates in their synthesis; compositions; formulations and methods, including methods of treating diseases, disorders or conditions, for example, cancer, in humans.

CROSS-REFERENCE

This application is a U.S. National Stage Application under 35 U.S.C. §371 of International Patent Application No. PCT/US2014/041414, filedJun. 6, 2014, which claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/832,132, filed Jun. 6, 2013, the content ofeach of which is incorporated herein by reference in its entirety.

FIELD

Provided herein are modified antibodies, compounds used to make them,and intermediates in their synthesis; compositions; formulations andmethods, including methods of treating diseases, disorders orconditions, for example, cancer, in humans.

BACKGROUND

Antibody cysteines can be conjugated to maleimides or other thiolspecific functional groups. Antibodies contains multiple interchaindisulfide bonds (e.g., 2 between the heavy chains and 2 between heavyand light chains for human IgG1 and IgG4) that covalently bond the heavyand light chains together and contribute to the stability of theantibodies in vivo. These interchain disulfides can be selectivelyreduced with dithiothreitol, tris(2-carboxyethyl)phosphine, or othermild reducing agents to afford 8 reactive sulfhydryl groups forconjugation.

Schumacher et al., “In Situ Maleimide Bridging of Disulfides and a NewApproach to Protein PEGylation”, Bioconjugate Chem. 2011, 22, 132-136,disclose the synthesis of 3,4-disubstituted maleimides such as3,4-bis(2-hydroxyethylsulfanyl)pyrrole-2,5-dione [referred to bySchumacher et al. as “dimercaptoethanolmaleimide”] and3,4-bis(phenylsulfanyl)pyrrole-2,5-dione [“dithiophenolmaleimide”], andtheir N-PEGylated derivatives as PEGylating agents for somatostatin,where the substituted maleimide bonds to the two sulfur atoms of thethiols of reduced cysteine-cysteine disulfide bond.

It would be desirable to develop modified antibodies, including thosethat do not compromise and/or enhance antibody stability.

SUMMARY

The present disclosure provides modified antibodies, compounds used tomake them, and intermediates in their synthesis; compositions;formulations and methods, including methods of treating diseases,disorders or conditions, for example, cancer, in humans.

In one aspect, provided herein is a method of modifying an antibody byreacting the antibody with a compound comprising a moiety of thefollowing formula (I):

or an enantiomer, diasteriomer, or mixtures thereof;wherein:

-   each Y and Y′ is independently hydrogen or an electrophilic leaving    group that reacts selectively with thiols, provided if one of Y and    Y′ is hydrogen, the other is the electrophilic leaving group;-   the    bond represents a single or a double bond; and-   the symbol    represents a point of attachment to another group;    wherein the antibody is modified by reacting the thiols of two    cysteine residues from at least one reduced interchain    cysteine-cysteine disulfide with the compound.

In certain embodiments of the method, the compound has the followingformula (Ia):

wherein:

-   each R and R′ is independently hydrogen or C₁₋₆ alkyl, wherein one    or more carbons in the C₁₋₆ alkyl are optionally substituted by a    group selected from an oxo, a thio, an imine, and a substituted    imine; or-   R and R′, together with the two carbons from the single or double    bond to which they are attached, form a saturated or unsaturated    carbocyclic ring containing from four to seven ring atoms; wherein,    excluding the two carbons from the single or double bond, one or    more of the ring carbon atoms are optionally replaced by a    heteroatom selected from O and N; wherein, excluding the two carbons    from the single or double bond, one or more of the ring carbon atoms    are optionally substituted by a group selected from an oxo, a thio,    an imine, a substituted imine, and a C₁₋₃ alkyl; and wherein, if    present, the ring nitrogen atom is optionally substituted by a C₁₋₃    alkyl.

In certain embodiments of the method, the moiety has one of thefollowing formulas (Ib) and (Ic):

or an enantiomer, diasteriomer, or mixtures thereof;wherein:

-   each X and X′ is independently absent, O, S, NH, or NR¹ wherein R¹    is C₁₋₃ alkyl;-   W is —O—, ═N—, or ═CH—; and-   each k and k′ is independently an integer of 0, 1, or 2.

In certain embodiments of the method, the compound has one of thefollowing formulas (Id) and (Ie):

wherein:

-   each R_(a), R_(a)′ and R_(b) is independently hydrogen, C₁₋₃alkyl,    or absent.

In certain embodiments of the method, the

bond represents a single bond.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of a halo, a substituted thiol, and asubstituted sulfonate. In certain embodiments, each Y and Y′ isindependently selected from the group consisting of chloro, bromo,fluoro, and iodo. In certain embodiments, each Y and Y′ is independentlyselected from an optionally substituted thiophenyl, an optionallysubstituted thionaphthyl, an optionally substituted thiopyridyl, anoptionally substituted isoquinoline, and an optionally substitutedphenylsulfonate.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of:

whereinR_(A) is selected from the group consisting of hydroxyl, amino, nitro,cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆ alkoxycarbonyl,C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₁₋₆alkoxy.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of:

In another aspect, provided herein is a modified antibody of thefollowing formula (II):

wherein:A is an antibody or antibody fragment;the two depicted cysteine residues are from at least one reducedinterchain cysteine-cysteine disulfide bond in A;n is an integer from 1 to 13;the

bond represents a single or a double bond; andthe symbol

represents a point of attachment to another group.

In certain embodiments, the modified antibody has the following formula(IIa):

wherein:

-   each R and R′ is independently hydrogen or C₁₋₆ alkyl, wherein one    or more carbons in the C₁₋₆ alkyl are optionally substituted by a    group selected from an oxo, a thio, an imine, and a substituted    imine; or-   R and R′, together with the two carbons from the single or double    bond to which they are attached, form a saturated or unsaturated    carbocyclic ring containing from four to seven ring atoms; wherein,    excluding the two carbons from the single or double bond, one or    more of the ring carbon atoms are optionally replaced by a    heteroatom selected from O and N; wherein, excluding the two carbons    from the single or double bond, one or more of the ring carbon atoms    are optionally substituted by a group selected from an oxo, a thio,    an imine, a substituted imine, and a C₁₋₃ alkyl; and wherein, if    present, the ring nitrogen atom is optionally substituted by a C₁₋₃    alkyl.

In certain embodiments, the modified antibody has one of the followingformulas (IIb) and (IIc):

wherein:

-   each X and X′ is independently absent, O, S, NH, or NR¹ wherein R¹    is C₁₋₃ alkyl;-   W is —O—, ═N—, or ═CH—; and-   each k and k′ is independently an integer of 0, 1, or 2.

In certain embodiments, the modified antibody has one of the followingformulas (IId) and (IIe):

wherein:

-   each R_(a), R_(a)′ and R_(b) is independently hydrogen, C₁₋₃alkyl,    or absent.

In certain embodiments of the modified antibody, the

bond represents a single bond.

In certain embodiments of the modified antibody, A is an antibody thatis specific to a cancer antigen. In certain embodiments, A is selectedfrom the group consisting of alemtuzumab, bevacizumab, brentuximab,cetuximab, gemtuzumab, ipilimumab, ofatumumab, panitumumab, rituximab,tositumomab, inotuzumab, glembatumumab, lovortuzumab and trastuzumab.Additional antibodies include adecatumumab, afutuzumab, bavituximab,belimumab, bivatuzumab, cantuzumab, citatuzumab, cixutumumab,conatumumab, dacetuzumab, elotuzumab, etaracizumab, farletuzumab,figitumumab, iratumumab, labetuzumab, lexatumumab, lintuzumab,lucatumumab, mapatumumab, matuzumab, milatuzumab, necitumumab,nimotuzumab, olaratumab, oportuzumab, pertuzumab, pritumumab,ranibizumab, robatumumab, sibrotuzumab, siltuximab, tacatuzumab,tigatuzumab, tucotuzumab, veltuzumab votumumab, and zalutumumab.

In another aspect, provided herein is an antibody or a formulationthereof obtainable by the methods disclosed herein.

In another aspect, provided herein is a process, which comprises:

(i) reducing one or more interchain cysteine-cysteine disulfide bonds inan antibody with a reducing agent; and

(ii) reacting the free thiol groups from the one or more reducedinterchain cysteine-cysteine disulfide bonds with a compound disclosedherein, thus producing a modified antibody.

In certain embodiments of the process, cysteine-cysteine disulfide bondsare reduced (e.g., 1 to 13 interchain disulfide bonds for IgG1, IgG2,IgG3, and/or IgG4). In certain embodiments of the process, for example,for IgG1, 1 to 4 interchain cysteine-cysteine disulfide bonds arereduced.

In certain embodiments of the process, the reducing agent isdithiothreitol (DTT) or tris(2 carboxyethyl)phosphine (TCEP).

In another aspect, provided herein is a pharmaceutical formulationcomprising a modified antibody disclosed herein.

In another aspect, provided herein is a method of treating a cancer byadministering to a human suffering therefrom a prophylactic ortherapeutically effective amount of a pharmaceutical formulationcomprising a modified antibody disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Human IgG Sub-types

FIG. 2: Forms of Antibodies and Antibody Fragments

FIG. 3: List of Antibodies in Clinical Trials

FIG. 4: Bifunctional Linkers for Improving Antibody Stability

FIG. 5: Antibody Only; RT=7.12

FIG. 6: Antibody+dibromosuccinimide−RT=7.24

FIG. 7: Antibody+dibromo-N-benzyl succinimide−RT=7.58

DETAILED DESCRIPTION

The present disclosure provides modified antibodies, compounds used tomake them, and intermediates in their synthesis; compositions;formulations and methods, including methods of treating diseases,disorders or conditions, for example, cancer, in humans. For example,the present disclosure provides modified antibodies and methods ofpreparing the modified antibodies, for example, by reacting the twothiol groups from at least one reduced interchain disulfide bond with acompound disclosed herein. Such chemically modified antibodies includethose that do not compromise and/or enhance antibody stability and areuseful in compositions and formulations, and in methods for treatingdiseases, disorders or conditions in humans.

Definitions

An “antibody,” also known as an immunoglobulin, is a large Y-shapedprotein that binds to an antigen. Antibodies are used by the immunesystem to identify and neutralize foreign objects such as bacteria andviruses. The antibody recognizes a unique part of the antigen, becauseeach tip of the “Y” of the antibody contains a site that is specific toa site on an antigen, allowing these two structures to bind withprecision. An antibody may consist of four polypeptide chains, two heavychains and two light chains connected by interchain cysteine disulfidebonds (see, e.g., FIG. 1). A “monoclonal antibody” is a monospecificantibody where all the antibody molecules are identical because they aremade by identical immune cells that are all clones of a unique parentcell. Initially, monoclonal antibodies are typically prepared by fusingmyeloma cells with the spleen cells from a mouse (or B-cells from arabbit) that has been immunized with the desired antigen, then purifyingthe resulting hybridomas by such techniques as affinity purification.Recombinant monoclonal antibodies are prepared in viruses or yeast cellsrather than in mice, through technologies referred to as repertoirecloning or phage display/yeast display, the cloning of immunoglobulingene segments to create libraries of antibodies with slightly differentamino acid sequences from which antibodies with desired specificitiesmay be obtained. The resulting antibodies may be prepared on a largescale by fermentation. “Chimeric” or “humanized” antibodies areantibodies containing a combination of the original (usually mouse) andhuman DNA sequences used in the recombinant process, such as those inwhich mouse DNA encoding the binding portion of a monoclonal antibody ismerged with human antibody-producing DNA to yield a partially-mouse,partially-human monoclonal antibody. Full-humanized antibodies areproduced using transgenic mice (engineered to produce human antibodies)or phage display libraries. Antibodies (Abs) and “immunoglobulins” (Igs)are glycoproteins having similar structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which generally lack antigen specificity. Polypeptides ofantibody-like molecules are produced at low levels by the lymph systemand at increased levels by myelomas. The terms “antibody” and“immunoglobulin” are used interchangeably in the broadest sense andinclude monoclonal antibodies (e.g., full length or intact monoclonalantibodies), polyclonal antibodies, monovalent antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity). Forms ofantibodies and antibody fragments are shown schematically in FIG. 2.These antibodies may also include certain antibody fragments. Anantibody can be chimeric, human, humanized and/or affinity matured.Exemplary antibodies are shown in FIG. 3. Antibodies of particularinterest are those that are specific to cancer antigens, arenon-immunogenic, have low toxicity, and are readily internalized bycancer cells; and suitable antibodies include alemtuzumab, bevacizumab,brentuximab, cetuximab, gemtuzumab, ipilimumab, ofatumumab, panitumumab,rituximab, tositumomab, inotuzumab, glembatumumab, lovortuzumab andtrastuzumab. Additional antibodies include adecatumumab, afutuzumab,bavituximab, belimumab, bivatuzumab, cantuzumab, citatuzumab,cixutumumab, conatumumab, dacetuzumab, elotuzumab, etaracizumab,farletuzumab, figitumumab, iratumumab, labetuzumab, lexatumumab,lintuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab,necitumumab, nimotuzumab, olaratumab, oportuzumab, pertuzumab,pritumumab, ranibizumab, robatumumab, sibrotuzumab, siltuximab,tacatuzumab, tigatuzumab, tucotuzumab, veltuzumab votumumab, andzalutumumab.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, and are not antibody fragments as definedbelow. The terms particularly refer to an antibody with heavy chainsthat contain the Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, two, three and as many as mostor all of the functions normally associated with that portion whenpresent in an intact antibody. In one aspect, an antibody fragmentcomprises an antigen binding site of the intact antibody and thusretains the ability to bind antigen. In another aspect, an antibodyfragment, such as an antibody fragment that comprises the Fc region,retains at least one of the biological functions normally associatedwith the Fc region when present in an intact antibody. Such functionsmay include FcRn binding, antibody half life modulation, ADCC functionand complement binding. In another aspect, an antibody fragment is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For example, such an antibody fragment maycomprise on antigen binding arm linked to an Fc sequence capable ofconferring in vivo stability to the fragment.

The term “monoclonal antibody,” as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. The modifier term “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain aspects, such a monoclonal antibody mayinclude an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones, or recombinant DNA clones. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. (See, Kohler et al., Nature, 256: 495 (1975); Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2.sup.nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981)), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, WO98/24893; WO96/34096; WO96/33735 andWO91/10741). The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567). “Humanized” formsof non-human (e.g., murine) antibodies are chimeric antibodies thatcontain minimal sequence derived from non-human immunoglobulin. In oneaspect, a humanized antibody is a human immunoglobulin (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such as mouse, rat, rabbit, or nonhuman primatehaving the desired specificity, affinity, and/or capacity. In anotheraspect, framework region (FR) residues of the human immunoglobulin arereplaced by corresponding non-human residues. In general, a humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable loops correspond to those of a non-human immunoglobulin,and all or substantially all the FRs are those of a human immunoglobulinsequence. The humanized antibody may comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See Vaswani and Hamilton, Ann. Allergy, Asthma &Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433(1994).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues. “Fc receptor” or “FcR” is areceptor that binds to the Fc region of an antibody. In certainembodiments, an FcR is a native human FcR. In one aspect, an FcR is onewhich binds an IgG antibody (a gamma receptor) and includes receptors ofthe Fc

RI, Fc

RII and Fc

RIII subclasses. (See Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).

An “antibody-drug conjugate” (ADC) is an antibody that is conjugated toone or more cytotoxins, through one or more linkers. The antibody istypically a monoclonal antibody specific to a therapeutic target such asa cancer antigen.

A “cytotoxic agent” or “cytotoxin” is a molecule that has a cytotoxiceffect on cells (e.g., when released within a cancer cell, is toxic tothat cell).

The term “linker,” as used herein, refers to a group of atoms used toconnect interconnecting moieties, for example, between an antibody andone or more cytotoxins in an ADC.

The term “polyether,” as used herein, refers to a polymer of alkylenethat contains at least one ether group, for example, polyoxyalkylene.

The term “polyethylene glycol” (PEG), as used herein, refers to apolymer of ethylene oxide having repeat units of —(CH₂CH₂—O)—.

The term “PEGylated linker,” as used herein, refers to a linkercomprising PEG. Examples of linkers include, for example, repeatingunits of —(CH₂CH₂O)_, —(CH₂CH₂O)CH₂CH₂—, —CH₂CH₂—(CH₂CH₂O)—, etc.

The term “alkyl,” as used herein, means a straight, branched chain, orcyclic (in this case, it would also be known as “cycloalkyl”)hydrocarbon containing from 1-10 carbon atoms. Examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylhexyl, n-heptyl, n-octyl,n-nonyl, and n-decyl. In certain embodiments, alkyl groups areoptionally substituted.

The term “C₁₋₁₀alkyl,” as used herein, means a straight, branched chain,or cyclic (in this case, it would also be known as “cycloalkyl”)hydrocarbon containing from 1-10 carbon atoms.

The term “C₁₋₆alkyl,” as used herein, means a straight, branched chain,or cyclic (in this case, it would also be known as “cycloalkyl”)hydrocarbon containing from 1-6 carbon atoms.

The term “C₁₋₃alkyl,” as used herein, means a straight or branched chainhydrocarbon containing from 1-3 carbon atoms.

The term “alkenyl,” as used herein, means a straight, branched chain, orcyclic (in which case, it would also be known as a “cycloalkenyl”)hydrocarbon containing from 2-10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens. Insome embodiments, depending on the structure, an alkenyl group is amonoradical or a diradical (e.g., an alkenylene group). In someembodiments, alkenyl groups are optionally substituted. Examples ofalkenyl include, but are not limited to, ethenyl, 2-propenyl,2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, and2-methyl-1-heptenyl. In certain embodiments, alkenyl groups areoptionally substituted.

The term “alkoxy,” as used herein, means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Examples of alkoxy include, but are not limited to, methoxy, ethoxy,propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

The term “C₂₋₆ alkenyl,” as used herein, means a straight, branchedchain, or cyclic (in this case, it would also be known as “cycloalkyl”)hydrocarbon containing from 2-6 carbon atoms and at least onecarbon-carbon double bond formed by the removal of two hydrogens.

The term “oxo,” as used herein, refers to the radical ═O. For example,in certain embodiments, a carbon atom substituted with an oxo group is

where the symbol

represents a point of attachment to another group.

The term “thio,” used herein, refers to the radical ═S. In certainembodiments, a carbon atom substituted with an thio group is

where the symbol

represents a point of attachment to another group.

The term “imine” as used herein, refers to the radical ═NH. In certainembodiments, a carbon atom substituted with an imine group is

where the symbol

represents a point of attachment to another group.

The term “substituted imine” as used herein, refers to the radical═N(R), wherein R is a substituent, for example, alkyl. In certainembodiments, a carbon atom substituted with a substituted imine group is

where the symbol

represents a point of attachment to another group.

The term “amino,” as used herein, refers to the radical —NH₂.

The term “hydroxyl” or “hydroxy,” as used herein, refers to the radical—OH.

The term “carboxyl” as used herein, refers to the radical —CO₂H.

The term “thiol,” as used herein, refers to the radical —SH. In certainembodiments, thiol refers to the side chain thiol group of a cysteineresidue.

The term “substituted thiol,” as used herein, refers to a radical suchas —SR wherein R is any optionally substituted chemical group describedherein. In certain embodiments, “substituted thiol” refers to a radical—SR where R is an alkyl, cycloalkyl, aryl or heteroaryl group as definedherein that may be optionally substituted as defined herein.Representative examples of substituted thiol include, but are notlimited to, thiophenyl, thionaphthyl, thiopyridyl, thioisoquinolinyl, asdepicted below:

The term “sulfonate,” as used herein, refers to the radical —OS(O₂)H.“Substituted sulfonate” refers to a radical such as —OS(O₂)R wherein Ris an alkyl, cycloalkyl, aryl or heteroaryl group as defined herein thatmay be optionally substituted as defined herein. In certain embodiments,R is selected from lower alkyl, alkyl, aryl and heteroaryl.Representative examples of substituted sulfonate include, but are notlimited to, tosylate, mesylate and triflate, as depicted below:

The term “carbocyclic group,” “carbocyclic ring,” or “carbocycle,” asused herein, refers to a hydrocarbon ring or fused ring systemcontaining from 3 to 14 ring atoms, and being fully saturated, or havingone or more double bonds between the ring atoms. In certain embodiments,the ring or fused ring system is fully saturated, in which case thecarbocycle is a “cycloalkyl group” or “cycloalkyl,” as defined herein.In certain embodiments, one or more ring carbon atoms is replaced by aheteroatom, for example, oxygen, sulfur and nitrogen, in which case thecarbocycle is a “heterocyclic group,” “heterocyclic ring,” or“heterocycle.” In certain embodiments, the carbocyclic group is anunsaturated aromatic carbocyclic group, which case the carbocycle is an“aryl group” or “aryl,” as defined herein. In certain embodiments, thecarbocyclic group is an unsaturated aromatic heterocyclic group, whichcase the carbocycle is an “heteroaryl group” or “heteroaryl,” as definedherein.

The term “aryl,” as used herein, refers to an unsaturated aromaticcarbocyclic group of from 6 to 14 carbon atoms having a single ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).Preferred aryls include phenyl, biphenyl, naphthyl and the like. Unlessotherwise constrained by the definition for the individual substituent,such aryl groups can optionally be substituted with 1 or moresubstituents, for example, 1 to 5 substituents, such as, hydroxyl,amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₁₋₆alkoxy, and the like.In certain embodiments, such aryl groups can optionally be substitutedwith 1 to 5 substituents selected from the group consisting of halo,cyano, nitro, CF₃—, CF₃O—, CH₃O—, —CO₂H, —C(O)CH₃, —NH₂, —OH, —SH,—NHCH₃, —N(CH₃)₂, —SMe and C₁₋₃ alkyl.

The term “heteroaryl,” as used herein, refers to an aryl ring systemhaving one to four heteroatoms as ring atoms in a heteroaromatic ringsystem, wherein the remainder of the atoms are carbon atoms. Suitableheteroatoms include oxygen, sulfur and nitrogen. Preferably, theheterocyclic ring system is monocyclic or bicyclic. Unless otherwiseconstrained by the definition for the individual substituent, suchheteroaryl groups can optionally be substituted with 1 or moresubstituents, for example, 1 to 5 substituents, such as, hydroxyl,amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₁₋₆ alkoxy, and the like.In certain embodiments, such heteroaryl groups can optionally besubstituted with 1 to 5 substituents selected from the group consistingof halo, cyano, nitro, CF₃—, CF₃O—, CH₃O—, —CO₂H, —C(O)CH₃, —NH₂, —OH,—SH, —NHCH₃, —N(CH₃)₂, —SMe and C₁₋₃ alkyl. Examples of hetroarylinclude, but are not limited to:

where Q is O, NR² or S.

The term “leaving group,” as used herein, refers to any group thatleaves in the course of a chemical reaction involving the group asdescribed herein and includes but is not limited to halogen, sulfonates(brosylate, mesylate, tosylate triflate etc. . . . ), p-nitrobenzoateand phosphonate groups, for example.

The term “electrophilic leaving group,” as used herein, refers to aleaving group that accepts an electron pair to make a covalent bond. Ingeneral, electrophiles are susceptible to attack by complementarynucleophiles, including the reduced thiols from the disulfide bond of anantibody.

The term “electrophilic leaving group that reacts selectively withthiols,” as used herein, refers to electrophilic leaving group thatreacts selectively with thiols, over other nucleophiles. In certainembodiments, an electrophilic leaving group that reacts selectively withthiols reacts selectively with the reduced thiols from the disulfidebond of an antibody.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one aspect, the cell-proliferative disorder is cancer.

“Tumor,” refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,”“proliferative disorder” and “tumor” are not mutually exclusive. Theterms “cancer” and “cancerous” refer to the physiological condition inmammals that is typically characterized by unregulated cell growth.Examples of cancer include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma and leukemia or lymphoid malignancies.

A “therapeutically effective amount” means that amount of a modifiedantibody, composition, or formulation disclosed herein which, whenadministered to a human suffering from a cancer, is sufficient to effecttreatment for the cancer. “Treating” or “treatment” of the cancerincludes one or more of:

(1) limiting/inhibiting growth of the cancer, e.g., limiting itsdevelopment;

(2) reducing/preventing spread of the cancer, e.g., reducing/preventingmetastases;

(3) relieving the cancer, e.g., causing regression of the cancer,

(4) reducing/preventing recurrence of the cancer; and

(5) palliating symptoms of the cancer.

Cancers of interest for treatment include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g., epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, oral cancer, liver cancer, bladdercancer, cancer of the urinary tract, hepatoma, breast cancer including,for example, HER2-positive breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia(CML), multiple myeloma and B-cell lymphoma, brain cancer, head and neckcancers and associated metastases.

Abbreviations/Acronyms

DPBS: Dulbecco's phosphate-buffered saline; DTPA:diethylenetriaminepentaacetic acid; DTT: dithiothreitol; PBS:phosphate-buffered saline; PEG: poly(ethyleneglycol); and TCEP:tris(2-carboxyethyl)phosphine.

Abbreviations/Acronyms (Antibodies)

ATZ: alemtuzumab; ATM: anitumumab; BCZ: bevacizumab; BTX: brentuximab;CTX: cetuximab; GTZ: gemtuzumab; GBT: glembatumumab; ITZ: inotuzumab;ILM: ipilimumab; LVT: lovortumumab; MTZ: milatuzumab; OTM: ofatumumab;RTX: rituximab; TTM: tositumomab; and TTZ: trastuzumab.

Compounds

The compounds used to make the modified antibodies disclosed herein maybe prepared by methods well known to a person of ordinary skill in theart, following procedures described in such references as Fieser andFieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley andSons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols.1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions,vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.:Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York,N.Y.; and Larock: Comprehensive Organic Transformations, VCH Publishers,New York, 1989. In some instances, the compounds used to make themodified antibodies disclosed herein may be purchased from commercialsources.

The following illustrate various embodiments of the compounds disclosedherein, which may be synthesized by these methods:

The above embodiments are merely illustrative, and not meant to belimiting.

Modified Antibodies

Compounds that coordinate to cysteine thiols of the antibody haveemployed monofunctional compounds, of which maleimide is an example.Reduction and opening of the cysteine-cysteine disulfide bonds to givefree thiols for conjugation decreases the stability of the antibody, andthe formation of the modified antibody by reaction of the reduced thiolsdoes not re-form the interchain disulfide bond, as illustrated in thefollowing Scheme A:

As a result, a heterogenous mixture of modified is produced, which maycomprise all possible positional isomers of conjugated compound tocysteine thiol, and may comprise all possible compound antibody ratios(1, 2, 3, 4, . . . , and 8).

In contrast, the compounds disclosed herein contain two reactivefunctional groups (e.g., Y and Y′ in the scheme below) that selectivelytarget the two sulfur atoms of the thiols of a reduced cysteine-cysteinedisulfide bond (e.g., interchain disulfide bond), as illustrated below:

Reaction of the compound with the two cysteine thiols gives a modifiedantibody conjugate with one compound per disulfide (e.g., one or moreinterchain disulfides) connected through two thioether bonds, asdepicted in FIG. 4 and shown in the following exemplary Scheme B:

Homogeneous modified antibodies are produced, for example, with acompound to antibody ratio of 4. Scheme B depicts a homogenous modifiedantibody, where, for example, the four (4) interchain disulfide bonds ofthe antibody (e.g., 2 H—H disulfide bonds, and 2 H-L disulfide bonds)are conjugated.

Unlike conventional methods for cysteine conjugation, the reactionre-forms a covalently bonded structure between the 2 cysteine sulfuratoms and therefore does not compromise and/or may enhance the overallstability of the antibody. The overall result is replacement of arelatively labile disulfide bond (e.g., interchain disulfide bond) witha stable “staple” or “snap” between the cysteines. The monosubstitutedcompounds (where one of Y and Y′ is hydrogen) are also effectivelybifunctional in conjugation with the antibody because the double bond iscapable of conjugation to one of the cysteine sulfur atoms and the Ygroup with the other.

Preparation of the Disclosed Compounds

The compounds disclosed herein may be prepared by methods well known toa person of ordinary skill in the art, following procedures described insuch references as Fieser and Fieser's Reagents for Organic Synthesis,vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistryof Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers,1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York,N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wileyand Sons, New York, N.Y.; and Larock: Comprehensive OrganicTransformations, VCH Publishers, New York, 1989.

Preparation of Modified Antibodies

Antibodies, typically monoclonal antibodies, are selected for binding toa specific antigen (e.g., cancer target), and purified andcharacterized. Therapeutic modified antibodies are prepared by standardmethods for cysteine conjugation, such as by methods analogous to thoseof Hamblett et al., “Effects of Drug Loading on the Antitumor Activityof a Monoclonal Antibody Drug Conjugate”, Clin. Cancer Res. 2004, 10,7063-7070; Doronina et al., “Development of potent and highlyefficacious monoclonal antibody auristatin conjugates for cancertherapy”, Nat. Biotechnol., 2003, 21(7), 778-784; and Francisco et al.,“cAC10-vcMMAE, an anti-CD30-monomethylauristatin E conjugate with potentand selective antitumor activity”, Blood, 2003, 102, 1458-1465. Modifiedantibody with four compounds per antibody are prepared by partialreduction of the antibody with an excess of a reducing reagent such asDTT or TCEP at 37° C. for 30 min, then the buffer exchanged by elutionthrough SEPHADEX® G-25 resin with 1 mM DTPA in DPBS. The eluent isdiluted with further DPBS, and the thiol concentration of the antibodymay be measured using 5,5′-dithiobis(2-nitrobenzoic acid) [Ellman'sreagent]. An excess, for example 5-fold, of the chemical compound isadded at 4° C. for 1 hr, and the conjugation reaction may be quenched byaddition of a substantial excess, for example 20-fold, of cysteine. Theresulting modified antibody mixture may be purified on SEPHADEX G-25equilibrated in PBS to remove unreacted compound, desalted if desired,and purified by size-exclusion chromatography. The resulting modifiedantibody may then be then sterile filtered, for example, through a 0.2μM filter, and lyophilized if desired for storage.

The formation of a modified antibody is described herein. illustrated byScheme B above depicts a “Y”-shaped structure denoting an antibody, forexample, an IgG1, where all four (4) interchain disulfide bonds of theantibody (2 H—H disulfide bonds, and 2 H-L disulfide bonds) are modifiedby conjugation with compound with a ratio of 4.

The Method:

In one aspect, provided herein is a method of modifying an antibody byreacting the antibody with a compound comprising a moiety of thefollowing formula (I):

or an enantiomer, diasteriomer, or mixtures thereof;wherein:

-   each Y and Y′ is independently hydrogen or an electrophilic leaving    group that reacts selectively with thiols, provided if one of Y and    Y′ is hydrogen, the other is the electrophilic leaving group;-   the    bond represents a single or a double bond; and-   the symbol    represents a point of attachment to another group.

In certain embodiments of the method, the antibody is modified byreacting the thiols of two cysteine residues from at least one reducedinterchain cysteine-cysteine disulfide with the compound.

In certain embodiments of the method, the

bond represents a single bond. In certain embodiments of the method, the

bond represents a double bond.

In certain embodiments of the method, the groups to which the moiety isattached do not include a polyether, for example, a PEG. In certainembodiments, the groups to which the moiety is attached do not include acytotoxin. In certain embodiments, the groups to which the moiety isattached do not include a linker, for example, a PEGylated linker. Incertain embodiments, the groups to which the moiety is attached do notinclude a linker conjugated to a cytotoxin.

In certain embodiments of the method, the compound has the followingformula (Ia):

or an enantiomer, diasteriomer, or mixtures thereof;wherein:

-   each R and R′ is independently hydrogen or C₁₋₆ alkyl, wherein one    or more carbons in the C₁₋₆ alkyl are optionally substituted by a    group selected from an oxo, a thio, an imine, and a substituted    imine; or-   R and R′, together with the two carbons from the single or double    bond to which they are attached, form a saturated or unsaturated    carbocyclic ring containing from four to seven ring atoms; wherein,    excluding the two carbons from the single or double bond, one or    more of the ring carbon atoms are optionally replaced by a    heteroatom selected from O and N; wherein, excluding the two carbons    from the single or double bond, one or more of the ring carbon atoms    are optionally substituted by a group selected from an oxo, a thio,    an imine, a substituted imine, and a C₁₋₃ alkyl; and wherein, if    present, the ring nitrogen atom is optionally substituted by a C₁₋₃    alkyl.

In certain embodiments of the method, the moiety has one of thefollowing formulas (Ib) and (Ic):

or an enantiomer, diasteriomer, or mixtures thereof;wherein:

-   -   each X and X′ is independently absent, O, S, NH, or NR¹ wherein        R¹ is C₁₋₃ alkyl;    -   W is —O—, ═N—, or ═CH—; and    -   each k and k′ is independently an integer of 0, 1, or 2.

In certain embodiments of the method, the compound has one of thefollowing formulas (Id) and (Ie):

wherein:

-   -   each R_(a), R_(a)′ and R_(b) is independently hydrogen,        C₁₋₃alkyl, or absent.

In certain embodiments of the method, where the moiety is of formula(Ib), k and k′ are both 0 (X and X′ are both absent), as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ib), k is 0 (X is absent), and k′ is 1, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ib), k is 1, and k′ is 0 (X′ is absent), as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ib), k and k′ are both 1, and each X and X′ is independently O or S; asdepicted below:

In certain embodiments of the method, where the moiety is of formula(Ib), k and k′ are both 1, and X and X′ are both absent, as depictedbelow:

In certain embodiments of the method, where the moiety is of formula(Ib), k and k′ are both 1, X is O or S, and X′ is absent, as depictedbelow:

In certain embodiments of the method, where the moiety is of formula(Ib), k and k′ are both 1, X is absent, and X′ is O or S, as depictedbelow:

Similarly, one of ordinary skill in the art will be able to envision allpossible embodiments of k, k′, X and X′ with regard to the moiety offormula (Ib).

In certain embodiments of the method, where the moiety is of formula(Ic), k is 0 (X is absent) and k′ is 2, as depicted below:

In certain subembodiments of the above embodiment, one or both of X′ mayalso absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 2 and k′ is 0 (X′ is absent), as depicted below:

In certain subembodiments of the above embodiment, one or both of X arealso absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k and k′ are both 1, and each X and X′ is independently O or S; asdepicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k and k′ are both 1, and X and X′ are both absent, as depictedbelow:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 1, k′ is 2, and X and X′ are both absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 2, k′ is 1, and X and X′ are both absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 2, k′ is 2, and X and X′ are both absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k and k′ are both 1, X is O or S, and X′ is absent, as depictedbelow:

In certain embodiments of the method, where the moiety is of formula(Ic), k and k′ are both 1, X is absent, and X′ is O or S, as depictedbelow:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 1, k′ is 2, X is O or S, and X′ is absent, as depicted below:

In certain embodiments of the method, where the moiety is of formula(Ic), k is 2, k′ is 1, X is absent, and X′ is O or S, as depicted below:

Similarly, one of ordinary skill in the art will be able to envision allpossible embodiments of k, k′, X and X′ with regard to the moiety offormula (Ic).

In certain embodiments of the method, each Y and Y′ is independentlyhydrogen or an electrophilic leaving group that reacts selectively withthiol.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of a halo, a substituted thiol, and asubstituted sulfonate. In certain embodiments, each Y and Y′ isindependently selected from the group consisting of chloro, bromo,fluoro, and iodo. In certain embodiments, each Y and Y′ is independentlyselected from an optionally substituted thiophenyl, an optionallysubstituted thionaphthyl, an optionally substituted thiopyridyl, anoptionally substituted isoquinoline, and an optionally substitutedphenylsulfonate.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of:

whereinR_(A) is selected from the group consisting of hydroxyl, amino, nitro,cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆ alkoxycarbonyl,C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₁₋₆alkoxy.

In certain embodiments of the method, each Y and Y′ is independentlyselected from the group consisting of:

The Antibody (A):

In certain embodiments, disclosed herein are antibodies and antibodyfragments (see, e.g., FIG. 2) for use in the methods disclosed herein.

In certain embodiments, A is an antibody or an antibody fragment. Incertain embodiments, A is a monoclonal antibody or monoclonal antibodyfragment.

In certain embodiments, the antibody (A) is a monoclonal antibody or ahumanized antibody. In certain embodiments, the antibody is specific toa cancer antigen. In another embodiment, the antibody employed in themodified antibodies disclosed herein is selected from the groupconsisting of alemtuzumab, bevacizumab, cetuximab, ipilimumab,ofatumumab, anitumumab, rituximab, tositumomab, inotuzumab,glembatumumab, lovortuzumab, milatuzumab and trastuzumab.

The Modified Antibody:

In another aspect, provided herein is a modified antibody of thefollowing formula (II):

wherein:A is an antibody or antibody fragment;the two depicted cysteine residues are from at least one reducedinterchain cysteine-cysteine disulfide bond in A;n is an integer from 1 to 13;the

bond represents a single or a double bond; andthe symbol

represents a point of attachment to another group.

In certain embodiments of the modified antibody, the

bond represents a single bond. In certain embodiments of the modifiedantibody, the

bond represents a double bond.

In certain embodiments, the modified antibody does not include an ADC.In certain embodiments, the modified antibody does not include anantibody with a linker (e.g., PEGylated linker). In certain embodiments,the modified antibody does not include an antibody conjugated to acytotoxin (e.g., a tubulin stabilizer, a tubulin destabilizer, a DNAalkylator, a DNA minor groove binder, a DNA intercalator, atopoisomerase I inhibitor, a topoisomerase II inhibitor, a gyraseinhibitor, a protein synthesis inhibitor, a proteosome inhibitor, ananti-metabolite, Actinomycin D, Amonafide, an auristatin, benzophenone,benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223),Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin,Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), amaytansinoid, Methotrexate, Mitoxantrone, Nocodazole, ProteosomeInhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol,a tubulysin, Velcade®, or Vincristine). In certain embodiments, themodified antibody does not include an antibody conjugated through alinker to a cytotoxin.

In certain embodiments of the modified antibody, the groups to which themoiety is attached do not include a polyether, for example, a PEG. Incertain embodiments, the groups to which the moiety is attached do notinclude a cytotoxin. In certain embodiments, the groups to which themoiety is attached do not include a linker, for example, a PEGylatedlinker. In certain embodiments, the groups to which the moiety isattached do not include a polyether (e.g., PEG) linker conjugated to acytotoxin.

In certain embodiments, the modified antibody has the following formula(IIa):

wherein:

-   each R and R′ is independently hydrogen or C₁₋₆ alkyl, wherein one    or more carbons in the C₁₋₆ alkyl are optionally substituted by a    group selected from an oxo, a thio, an imine, and a substituted    imine; or-   R and R′, together with the two carbons from the single or double    bond to which they are attached, form a saturated or unsaturated    carbocyclic ring containing from four to seven ring atoms; wherein,    excluding the two carbons from the single or double bond, one or    more of the ring carbon atoms are optionally replaced by a    heteroatom selected from O and N; wherein, excluding the two carbons    from the single or double bond, one or more of the ring carbon atoms    are optionally substituted by a group selected from an oxo, a thio,    an imine, a substituted imine, and a C₁₋₃ alkyl; and wherein, if    present, the ring nitrogen atom is optionally substituted by a C₁₋₃    alkyl.

In certain embodiments, the modified antibody has one of the followingformulas (IIb) and (IIc):

wherein:

-   each X and X′ is independently absent, O, S, NH, or NR¹ wherein R¹    is C₁₋₃ alkyl;-   W is —O—, ═N—, or ═CH—; and-   each k and k′ is independently an integer of 0, 1, or 2.

In certain embodiments, the modified antibody has one of the followingformulas (IId) and (IIe):

wherein:

-   each R_(a), R_(a)′ and R_(b) is independently hydrogen, C₁₋₃alkyl,    or absent.

In certain embodiments of the modified antibody, A is an antibody thatis specific to a cancer antigen. In certain embodiments, A is selectedfrom the group consisting of alemtuzumab, anitumumab, bevacizumab,brentuximab, cetuximab, gemtuzumab, glembatumumab, inotuzumab,ipilimumab, lovortumumab, milatuzumab, ofatumumab, rituximab,tositumomab, and trastuzumab.

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k and k′ are both 0 (X and X′ are both absent), asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k is 0 (X is absent), and k′ is 1, as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k is 1, and k′ is 0 (X′ is absent), as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k and k′ are both 1, and each X and X′ isindependently O or S; as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k and k′ are both 1, and X and X′ are both absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k and k′ are both 1, X is O or S, and X′ is absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIb), k and k′ are both 1, X is absent, and X′ is O or S, asdepicted below:

Similarly, one of ordinary skill in the art will be able to envision allpossible embodiments of k, k′, X and X′ with regard to the antibody offormula (IIb).

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 0 (X is absent) and k′ is 2, as depicted below:

In certain subembodiments of the above embodiment, one or both of X′ mayalso absent, as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 2 and k′ is 0 (X′ is absent), as depicted below:

In certain subembodiments of the above embodiment, one or both of X arealso absent, as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k and k′ are both 1, and each X and X′ isindependently O or S; as depicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k and k′ are both 1, and X and X′ are both absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 1, k′ is 2, and X and X′ are both absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 2, k′ is 1, and X and X′ are both absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 2, k′ is 2, and X and X′ are both absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k and k′ are both 1, X is O or S, and X′ is absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k and k′ are both 1, X is absent, and X′ is O or S, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 1, k′ is 2, X is O or S, and X′ is absent, asdepicted below:

In certain embodiments of the modified antibody, where the antibody isof formula (IIc), k is 2, k′ is 1, X is absent, and X′ is O or S, asdepicted below:

Similarly, one of ordinary skill in the art will be able to envision allpossible embodiments of k, k′, X and X′ with regard to the antibody offormula (IIc).

Assays

The antibodies disclosed herein may be assayed for binding affinity toand specificity for the desired antigen by any of the methodsconventionally used for the assay of antibodies; and they may be assayedfor efficacy as therapeutics. A person of ordinary skill in the art willhave no difficulty, considering that skill and the literature available,in determining suitable assay techniques; from the results of thoseassays, in determining suitable doses to test in humans as therapeuticagents, and, from the results of those tests, in determining suitabledoses to use to treat diseases, disorders or conditions in humans.

Formulation and Administration

The antibodies disclosed herein will typically be formulated assolutions for intravenous administration, or as lyophilized concentratesfor reconstitution to prepare intravenous solutions (to bereconstituted, e.g., with normal saline, 5% dextrose, or similarisotonic solutions). They will typically be administered by intravenousinjection or infusion. A person of ordinary skill in the art ofpharmaceutical formulation, especially the formulation of therapeuticantibodies, will have no difficulty, considering that skill and theliterature available, in developing suitable formulations.

EXAMPLES Synthesis of Compounds

The following procedures may be employed for the preparation of thecompounds disclosed herein. The starting materials and reagents used inpreparing these compounds are either available from commercial supplierssuch as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem(Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methodswell known to a person of ordinary skill in the art, followingprocedures described in such references as Fieser and Fieser's Reagentsfor Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y.,1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps.,Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, JohnWiley and Sons, New York, N.Y., 1991; March J.: Advanced OrganicChemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock:Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

In some cases, protective groups may be introduced and finally removed.Suitable protective groups for amino, hydroxy and carboxy groups aredescribed in Greene et al., Protective Groups in Organic Synthesis,Second Edition, John Wiley and Sons, New York, 1991. Standard organicchemical reactions can be achieved by using a number of differentreagents, for examples, as described in Larock: Comprehensive OrganicTransformations, VCH Publishers, New York, 1989.

Example 1: Antibody Modified with 1,2-dibromoethene

Trastuzumab, 1 mL of a 20 mg/mL solution in pH 7.4 PBS (Gibco Mg and Cafree) with 1 mM DTPA, is loaded into a sterile 1.7 mL Eppendorf tube,then 2.75 equivalents of TCEP hydrochloride (Sigma ampule 0.5Mconcentration), is added and the mixture incubated at 37° C. for 1 hourto give an average of 4 free thiol pairs per trastuzumab (this can beverified by Ellman's colorimetric assay—see Ellman, “Tissue sulfhydrylgroups”, Arch. Biochem. Biophys, 1959, 82, 70-77 or later papersreferring to this assay). The reduced antibody solution is cooled in anice-bath at about 0° C. for 15 minutes; then a solution of about 4.5equivalents of 1,2-dibromoethene in dimethylsulfoxide is added and themixture incubated at 37° C. for 2 hours (or at 4° C. for 20 hours). Theresulting modified antibody is purified by size-exclusion chromatography(GE ÄKTA pure chromatographic system) or PD10 desalting column.

Example 2: Antibody Modified with 1,2-dibromopentan-3-one

Trastuzumab, 1 mL of a 20 mg/mL solution in pH 7.4 PBS (Gibco Mg and Cafree) with 1 mM DTPA, is loaded into a sterile 1.7 mL Eppendorf tube,then 2.75 equivalents of TCEP hydrochloride (Sigma ampule 0.5Mconcentration), is added and the mixture incubated at 37° C. for 1 hourto give an average of 4 free thiol pairs per trastuzumab (this can beverified by Ellman's colorimetric assay—see Ellman, “Tissue sulfhydrylgroups”, Arch. Biochem. Biophys, 1959, 82, 70-77 or later papersreferring to this assay). The reduced antibody solution is cooled in anice-bath at about 0° C. for 15 minutes; then a solution of about 4.5equivalents of 1,2-dibromopentan-3-one in dimethylsulfoxide is added andthe mixture incubated at 37° C. for 2 hours (or at 4° C. for 20 hours).The resulting modified antibody is purified by size-exclusionchromatography (GE ÄKTA pure chromatographic system) or PD10 desaltingcolumn.

Example 3: Modified Antibody with1-methyl-3,4-bis(pyridin-2-ylthio)-1H-pyrrole-2,5-dione

Trastuzumab, 1 mL of a 20 mg/mL solution in pH 7.4 PBS (Gibco Mg and Cafree) with 1 mM DTPA, is loaded into a sterile 1.7 mL Eppendorf tube,then 2.75 equivalents of TCEP hydrochloride (Sigma ampule 0.5Mconcentration), is added and the mixture incubated at 37° C. for 1 hourto give an average of 4 free thiol pairs per trastuzumab (this can beverified by Ellman's colorimetric assay—see Ellman, “Tissue sulfhydrylgroups”, Arch. Biochem. Biophys, 1959, 82, 70-77 or later papersreferring to this assay). The reduced antibody solution is cooled in anice-bath at about 0° C. for 15 minutes; then a solution of about 4.5equivalents of 1-methyl-3,4-bis(pyridin-2-ylthio)-1H-pyrrole-2,5-dionein dimethylsulfoxide is added and the mixture incubated at 37° C. for 2hours (or at 4° C. for 20 hours). The resulting modified antibody ispurified by size-exclusion chromatography (GE ÄKTA pure chromatographicsystem) or PD10 desalting column.

Similar syntheses using other disclosed compounds, and/or otherantibodies, give the corresponding modified antibody.

As shown in FIGS. 5-7, the modified antibodies prepared from the methodsof the present application provide products with significant homogeneityas shown by HIC traces, when compared with modified antibodies preparedby conventional methods that provide inhomogeneous antibodies withmultiple products and positional isomers.

Assays

The modified antibodies disclosed herein are tested for potency andselectivity in vitro by determining their activity (e.g., cytotoxicityin cancer cell lines) of interest, such as those cancer cell linesexpressing the antigen corresponding to the antibody. The modifiedantibodies disclosed herein are tested for potency and safety in vivo inanimal models of therapeutic efficacy such as the mouse subcutaneouscancer xenograft and mouse orthotopic cancer xenograft models well knownto those of skill in the art of cancer research.

Example 4: Binding and Activity of Modified Antibody Compared toUnmodified Antibody

The activity of a modified antibody disclosed herein is compared to theactivity of the parental antibody (e.g., unmodified) for example,anti-tumor activity in HER2-positive and HER2-negative tumor cells. Incertain embodiments, the modified antibodies are as potent and/orstable, or more potent and/or more stable, than their parental (e.g.,unmodified) antibodies.

Example 5: Binding Affinity of Modified Antibodies forAntigen-Expressing Cells

Binding of the antibodies and modified antibodies to antigen-expressingcells are measured using a cell ELISA. Cells transduced to express thetarget antigen (e.g., sarcoma cells for HER2, CD98, C10orf54/VISTA) areplated the day at 5000 cells per well in a 384-well plate. The followingday, antibodies and modified antibodies are serially diluted in aseparate plate, and then transferred to the cell plate, which haspreviously had media removed by aspiration. After a 2 hour incubation atroom temperature, the plate is washed with wash buffer (DPBS at pH7.4with 0.1% bovine serum albumin) and then 25 μL horseradishperoxidase-labeled secondary antibody diluted in media is added andincubated for 30 minutes at room temperature. The plate is then washedand 15 μL of a chemiluminescent substrate (Pierce catalog #37069) isadded; and the plate is read in a plate-based luminescence reader.Antibodies and modified antibodies demonstrating comparable affinity forcells expressing the target antigen indicate that modification does notnegatively affect antigen binding.

Example 6: Potency of Modified Antibodies Against Antigen-ExpressingCells

The potency of the modified antibodies disclosed herein for inhibitionof tumor cell growth is tested in cell proliferation assays. The Ramos(B-cell lymphoma) Kasumi-3 (acute myeloid leukemia) and BT474 (HER2+human breast carcinoma) cell lines are seeded into 96 well half-areaplates the day before drug treatment at 3000 and 5000 cells per wellrespectively. Modified antibodies and controls are serially diluted in amaster plate, and then transferred to the cell plates, which areincubated at 37° C. and 5% CO₂ for 3 days. The cells are quantitated bymeasuring the level of ATP in the wells using the ATPLite 1 Step kit(Perkin Elmer catalog #50-904-9883) as described by the manufacturer.

Example 7: Efficacy of Modified Antibodies in Murine Xenograft Models

The Ramos Cell Xenograft Model.

The Ramos cell line is obtained from ATCC and cultured according to thesupplier's protocols. 4-6 Week-old immunodeficient female mice (TaconicC.B—17 scid) are subcutaneously injected on the right flank with 1×10⁷viable cells in a mixture of PBS (without magnesium or calcium) and BDMatrigel (BD Biosciences) at a 1:1 ratio. The injected total volume permouse is 200 μL with 50% being Matrigel. Once the tumor reaches a sizeof 65-200 mm³, mice are randomized. Modified antibodies are formulatedin PBS and administered once intravenously at a dose of 1 mg/Kg into thelateral tail vein, and body weights and tumors are measured twiceweekly. Tumor volume are calculated as described in van der Horst etal., “Discovery of Fully Human Anti-MET Monoclonal Antibodies withAntitumor Activity against Colon Cancer Tumor Models In vivo”,Neoplasia, 2009, 11, 355-364. The experiments are performed on groups of8 animals per experimental point. The negative control group receivesHB121 (an IgG2a-negative antibody) at a concentration equimolar to theconcentration that would be released by the modified antibody, while thepositive control group receives the modified antibody.

The BT474 Cell Xenograft Model.

The BT474 cell line is obtained from ATCC and cultured according to thesupplier's protocols. 4-6 Week-old immunodeficient female mice (TaconicC.B—17 scid) are implanted with a δ-estradiol pellet 3 days before beingsubcutaneously injected on the right flank with 1×10⁷ viable cells in amixture of PBS (without magnesium or calcium) and BD Matrigel (BDBiosciences) at a 1:1 ratio. The injected total volume per mouse is 200μL with 50% being Matrigel. Once the tumor reaches a size of 100-150mm³, mice are randomized. Modified antibodies are formulated in PBS andadministered once intravenously at a dose of 1 mg/Kg into the lateraltail vein, and body weights and tumors are measured twice weekly. Tumorvolume is calculated as described in van der Horst et al., cited above.The experiments are performed on groups of 8 animals per experimentalpoint. The negative control group receives HB121 at a concentrationequimolar to the concentration that would be released by the modifiedantibodies, while the positive control group receives trastuzumab at 1mg/Kg.

Similar tests are conducted with cell lines for other cancers (thoseexpressing different antigens) and modified antibodies where theantibody binds to the antigen expressed by the cancer.

While a number of exemplary embodiments, aspects and variations havebeen provided herein, those of skill in the art will recognize certainmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations. It isintended that the following claims are interpreted to include all suchmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations are withintheir scope.

What is claimed is:
 1. A method of modifying an antibody by reacting theantibody with a compound comprising a moiety of the following formula(Ia):

or an enantiomer, diastereomer, or mixtures thereof; wherein: each Y andY′ is independently selected from

wherein: each R and R′ is independently hydrogen or C₁₋₆ alkyl, whereinone or more carbons in the C₁₋₆ alkyl are optionally substituted by anoxo group; or R and R′, together with the two carbons from the single ordouble bond to which they are attached, form a saturated or unsaturatedcarbocyclic ring containing from four to seven ring atoms; wherein,excluding the two carbons from the single or double bond, one or more ofthe ring carbon atoms are optionally replaced by N; wherein, excludingthe two carbons from the single or double bond, one or more of the ringcarbon atoms are optionally substituted by an oxo group; and wherein, ifpresent, the ring nitrogen atom is substituted by a C₁₋₃ alkyl; the

bond represents a single or a double bond; and the symbol

represents a point of attachment to another group; wherein R_(A) isselected from the group consisting of hydroxyl, amino, nitro, cyano,chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₁₋₆ alkoxy; and wherein the antibody ismodified by reacting the thiols of two cysteine residues from at leastone reduced interchain cysteine-cysteine disulfide with the compound. 2.The method of claim 1, wherein the

represents a single bond.
 3. A process, which comprises: (i) reducingone or more interchain cysteine-cysteine disulfide bonds in an antibodywith a reducing agent; and (ii) reacting the free thiol groups from theone or more reduced interchain cysteine-cysteine disulfide bonds with acompound, thus producing a modified antibody; wherein the compoundcomprises a moiety of the following formula (la):

or an enantiomer, diastereomer, or mixtures thereof; wherein: each Y andY′ is independently selected from

wherein: each R and R′ is independently hydrogen or C₁₋₆ alkyl, whereinone or more carbons in the C₁₋₆ alkyl are optionally substituted by anoxo group; or R and R′, together with the two carbons from the single ordouble bond to which they are attached, form a saturated or unsaturatedcarbocyclic ring containing from four to seven ring atoms; wherein,excluding the two carbons from the single or double bond, one or more ofthe ring carbon atoms are optionally replaced by N; wherein, excludingthe two carbons from the single or double bond, one or more of the ringcarbon atoms are optionally substituted by an oxo group; and wherein, ifpresent, the ring nitrogen atom is optionally substituted by a C₁₋₃alkyl; the

represents a single or a double bond; and the symbol

represents a point of attachment to another group; wherein R_(A) isselected from the group consisting of hydroxyl, amino, nitro, cyano,chloro, bromo, fluoro, iodo, oxo, carboxyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₁₋₆ alkoxy; and wherein the antibody ismodified by reacting the thiols of two cysteine residues from at leastone reduced interchain cysteine-cysteine disulfide with the compound. 4.The process of claim 3, wherein 1 to 4 interchain cysteine-cysteinedisulfide bonds are reduced.
 5. The process of claim 3, wherein 4interchain cysteine-cysteine disulfide bonds are reduced.
 6. The processof claim 3, wherein the reducing agent is dithiothreitol (DTT) or tris(2carboxyethyl)phosphine (TCEP).
 7. The method of claim 1, wherein thebond represents a double bond.
 8. The method of claim 1, wherein each Yand Y′ is


9. The method of claim 1, wherein the compound is 1methyl-3,4-bis(pyridine-2-ylthio)-1H-pyrrole-2,5-dione.
 10. The methodof claim 1, wherein the moiety is represented by the following formulas(Ib) or (Ic):

or an enantiomer, diasteriomer, or mixtures thereof; wherein: each X andX′ is independently absent or O; W is ═N—; and each k and k′ isindependently an integer of 0, 1, or
 2. 11. The method of claim 10,wherein the moiety is represented by formula (Ib), and wherein: (a) kand k′ are both 0 and X and X′ are both absent as depicted below:

or (b) k is 0, X is absent and k′ is 1 as depicted below:

or (c) k is 1, k′ is 0 and X′ is absent as depicted below:

or (d) k and k′ are both 1 and X and X′ are both O as depicted below:

or (e) k and k′ are both 1 and X and X′ are both absent as depictedbelow:

or (f) k and k′ are both 1, X is O and X′ is absent as depicted below:

or (g) k and k′ are both 1, X is absent and X′ is O as depicted below:


12. The method of claim 10, wherein the moiety is represented by formula(Ic) and wherein: (a) k is 0, X is absent and k′ is 2 as depicted below:

or (b) k is 2, k′ is 0 and X′ is absent as depicted below:

or (c) k and k′ are both 1 and each X and X′ is O as depicted below:

or (d) k and k′ are both 1, and X and X′ are both absent as depictedbelow:

or (e) k is 1, k′ is 2 and X and X′ are both absent as depicted below:

or (f) k is 2, k′ is 1 and X and X′ are both absent as depicted below:

or (g) k is 2, k′ is 2 and X and X′ are both absent as depicted below:

or (h) k and k′ are both 1, X is O and X′ is absent, as depicted below:

or (i) k and k′ are both 1, X is absent and X′ is O as depicted below:

or (j) k is 1, k′ is 2, X is O and X′ is absent as depicted below:

or (k) k is 2, k′ is 1, X is absent and X′ is O as depicted below:


13. The method of claim 12, wherein in (a), one or both of X′ is alsoabsent as depicted below:


14. The method of claim 12, wherein in (b), one or both of X is alsoabsent as depicted below:


15. The method of claim 10, wherein the moiety is of the followingformulas (Id) or (le):

wherein: each R_(a), R_(a)′ and R_(b) is C₁₋₃ alkyl.
 16. The method ofclaim 1, wherein the moiety is selected from the group consisting of:

and.
 17. The method of claim 1, wherein the moiety is selected from thegroup consisting of:

and.
 18. The process of claim 3, wherein the

bond represents a single bond.
 19. The process of claim 3, wherein the

bond represents a double bond.
 20. The process of claim 3, wherein eachY and Y′ is


21. The process of claim 3, wherein the compound is1-methyl-3,4-bis(pyridine-2-ylthio)-1H-pyrrole-2,5-dione.
 22. Theprocess of claim 3, wherein the moiety is represented by the followingformulas (Ib) or (Ic):

or an enantiomer, diasteriomer, or mixtures thereof; wherein: each X andX′ is independently absent or O; W is ═N—; and each k and k′ isindependently an integer of 0, 1, or
 2. 23. The process of claim 22,wherein the moiety is represented by formula (Ib), and wherein: (a) kand k′ are both 0 and X and X′ are both absent as depicted below:

or (b) k is 0, X is absent and k′ is 1 as depicted below:

or (c) k is 1, k′ is 0 and X′ is absent as depicted below:

or (d) k and k′ are both 1 and X and X′ are both O as depicted below:

or (e) k and k′ are both 1 and X and X′ are both absent as depictedbelow:

or (f) k and k′ are both 1, X is O and X′ is absent as depicted below:

or (g) k and k′ are both 1, X is absent and X′ is O as depicted below:


24. The process of claim 22, wherein the moiety is represented byformula (Ic) and wherein: (a) k is 0, X is absent and k′ is 2 asdepicted below:

or (b) k is 2, k′ is 0 and X′ is absent as depicted below:

or (c) k and k′ are both 1 and each X and X′ is O as depicted below:

or (d) k and k′ are both 1, and X and X′ are both absent as depictedbelow:

or (e) k is 1, k′ is 2 and X and X′ are both absent as depicted below:

or (f) k is 2, k′ is 1 and X and X′ are both absent as depicted below:

or (g) k is 2, k′ is 2 and X and X′ are both absent as depicted below:

or (h) k and k′ are both 1, X is O and X′ is absent, as depicted below:

or (i) k and k′ are both 1, X is absent and X′ is O as depicted below:

or (j) k is 1, k′ is 2, X is O and X′ is absent as depicted below:

or (k) k is 2, k′ is 1, X is absent and X′ is O as depicted below:


25. The process of claim 24, wherein in (a), one or both of X′ is alsoabsent as depicted below:


26. The process of claim 24, wherein in (b), one or both of X is alsoabsent as depicted below:


27. The process of claim 22, wherein the moiety is represented by thefollowing formulas (Id) or (Ie):

wherein: each R_(a), R_(a)′ and R_(b) is C₁₋₃ alkyl.
 28. The process ofclaim 3, wherein the moiety is selected from the group consisting of:

and.
 29. The process of claim 3, wherein the moiety is selected from thegroup consisting of:

and.